Resiquimod r848

MCs from spleens and LNs were thawed, washed and resuspended in culture medium (RPMI 1640) supplemented with 10% FCS, l‐glutamine (2 mmol L⁻¹), penicillin G sodium (100 U mL⁻¹) and streptomycin sulphate (100 g mL⁻¹, all from Thermo Fisher Scientific) to a concentration of 10⁶ cells mL⁻¹. A proportion of MCs were activated with 10 ng mL⁻¹ phorbol 12‐myristate 13‐acetate (PMA) and 1 μg mL⁻¹ ionomycin (both from Merck, Stockholm) in the presence of protein transport inhibitor monensin (GolgiStop, BD Biosciences, Stockholm) for 4 h at 37°C and 5% CO₂, ahead of flow cytometric analysis of T‐cell subsets and their cytokines. MCs in culture medium with monensin or in culture medium alone served as nonstimulated controls or for immunophenotyping of B lymphocytes, respectively. To assess total and vaccine‐specific IgG‐secreting MBCs, MCs were pre‐activated with 1 μg mL⁻¹ resiquimod (R848) and 10 ng mL⁻¹ IL‐2 (both from Mabtech, Stockholm) in culture plates for 72 h. IL‐21‐secreting cells were enumerated after overnight activation of MCs with 10 ng mL⁻¹ PMA and 1 μg mL⁻¹ ionomycin directly into wells of pretreated and coated ELISPOT plates (details found below the ELISPOT heading). MCs in cell culture medium only served as negative controls in both cases. Following the activation steps and prior to ELISPOT, cell culture supernatants were collected and stored at −80°C.

MERS-CoV-S-specific B cells were analyzed using an antigen-specific IgG ELISpot. Cryopreserved PBMC were thawed, resuspended to 2×10⁶ cells/mL and stimulated in R10 containing 1% Hepes (Thermo Fisher Scientific), 0.5 μg/mL resiquimod (R848, Mabtech) and 5 ng/mL interleukin-2 (IL-2, Mabtech) for 75 h at 37°C and 5% CO₂. PVDF-Multi-Screen-IP plates (Millipore) were treated with 15 μL/well 35% ethanol and washed with sterile water. Plates were coated overnight at 4°C with 100 μL/well of either PBS containing anti-IgG capture antibody (15 μg/mL, Mabtech), MERS-CoV-S protein S1 or S2 subunit (10 μg/mL, SinoBiological), or PBS only. Plates were washed and after 30 min blocking with R10 containing 1% Hepes, pre-stimulated PBMC were added to two replicate wells of each coating condition and incubated for 16 h at 37°C and 5% CO_2. For detection of spots, biotinylated anti-IgG detection antibody, streptavidin-ALP and BCIP/NBT-plus substrate solution (Mabtech) were used according to the manufacturer’s protocol. Plates were analyzed using an AID EliSpot Reader System (AID GmbH). The cut-off value was set at 6.6 SFU, calculated as the geometric mean of blank wells +2 standard deviations.